請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54031
標題: | 探討Cyp11a1基因缺乏對小鼠生殖之影響 Effect of Cyp11a1 deficiency on mouse reproduction |
作者: | Shuo-Hui Hsu 徐碩惠 |
指導教授: | 胡孟君 |
關鍵字: | Cyp11a1基因缺乏,生殖, Cyp11a1 deficiency,reproduction, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 膽固醇側鏈裂解酶(cytochrome P450 side chain cleavage,P450scc)為Cyp11a1基因的產物,擔任類固醇賀爾蒙生成的速率決定步驟,能將膽固醇轉化為孕烯醇酮(pregnenolone)。然而,在小鼠動物模式當中,Cyp11a1基因經全面性剔除 (Cyp11a1-/-)之後會導致嚴重的腎上腺功能不足,小鼠出生不久即死亡,因此未能探究Cyp11a1基因在成鼠性腺中所扮演的角色。為了突破這個限制,我們使用Cre/loxP重組酶系統將性腺中的Cyp11a1基因選擇性剔除,此性腺Cyp11a1基因條件式剔除小鼠(Cyp11a1f/f;SCCmtp-Cre、Cyp11a1f/-;SCCmtp-Cre)能夠存活至成鼠並且擁有正常的外生殖器特徵。經西方墨點分析,Cyp11a1f/-;SCCmtp-Cre小鼠睪丸的P450scc蛋白質量與野生型Cyp11a1f/f 相比,下降了83.4%。雖然P450scc蛋白質量有大幅度的下降,但並不影響Cyp11a1f/-;SCCmtp-Cre小鼠精子形成及生殖功能。Cyp11a1f/f;SCCmtp-Cre與Cyp11a1f/-;SCCmtp-Cre小鼠卵巢組織的P450scc蛋白質量與Cyp11a1f/f 相比則是分別下降了96.5%和98.2%。Cyp11a1f/f;SCCmtp-Cre與Cyp11a1f/-;SCCmtp-Cre的卵巢組織切片,可以觀察到發育中各個時期的濾泡與黃體,但黃體以及卵巢基質細胞的細胞質中充滿空泡。經腎上腺移植存活的Cyp11a1-/- 成鼠,也有正常濾泡,但無黃體產生。Cyp11a1f/f;SCCmtp-Cre與Cyp11a1f/-;SCCmtp-Cre具有正常型態的子宮,但Cyp11a1-/- 雌鼠子宮發育不全。Cyp11a1f/-;SCCmtp-Cre 雌鼠擁有正常的性週期,於超排卵試驗能夠排出正常數量的卵,且受精卵在體外培養有能力發育至二細胞期。然而,Cyp11a1f/-;SCCmtp-Cre雌鼠無法懷孕,Cyp11a1f/f;SCCmtp-Cre雌鼠則具有生殖能力。根據上述結果可以歸納出,卵巢中P450scc蛋白質量多寡為子宮發育、排卵能力與生殖能力的關鍵性因素。 Cyp11a1 encodes cytochrome P450scc, the mitochondrial cholesterol side chain cleavage enzyme, is the only enzyme that catalyzes the conversion of cholesterol to pregnenolone and, thus, is required for the biosynthesis of all steroid hormones. Cyp11a1 knockout mice (Cyp11a1-/- ) died shortly after birth because of severe adrenal insufficiency; however, the roles of Cyp11a1 in the gonads remain unclear. In this study, we used a Cre/loxP approach to specifically knockout Cyp11a1 in gonads. Both male and female conditional knockout mice (Cyp11a1f/f;SCCmtp-Cre, Cyp11a1f/-;SCCmtp-Cre) survive to adulthood with normal gross external phenotype. Western blot analysis revealed that the protein levels of P450scc in testis are reduced by 83.4% in Cyp11a1f/-;SCCmtp-Cre mice compared to wild-type Cyp11a1f/f. Adult male Cyp11a1f/-;SCCmtp-Cre mice are fertile and have normal sperm formation in testis. In ovary, the protein levels of P450scc were reduced by 96.5% and 98.2% in Cyp11a1f/f;SCCmtp-Cre and Cyp11a1f/-;SCCmtp-Cre, respectively. Ovary of Cyp11a1f/f;SCCmtp-Cre and Cyp11a1f/-;SCCmtp-Cre mice displayed various stages of follicles and corpus luteum; however, the cytoplasmic vacuolation is found in luteal cells. The vacuolated cytoplasm was also present in the stromal cells in Cyp11a1f/f;SCCmtp-Cre and Cyp11a1f/-;SCCmtp-Cre ovary. Cyp11a1-/- mice can be rescued and survived to adulthood by adrenal transplantation. Cyp11a1-/- female appeared have normal follicles, but the corpus luteum was not found in the ovary. Cyp11a1f/f;SCCmtp-Cre and Cyp11a1f/-;SCCmtp-Cre female had normal uterus, however, the uterus of Cyp11a1-/- mice exhibited significant underdevelopment. The female Cyp11a1f/-;SCCmtp-Cre mice have normal estrus cycle and showed responses to superovulation treatment to produced fertilized eggs that can develop into 2-cell stage in vitro. However, Cyp11a1f/-;SCCmtp-Cre female mice are infertile. In contrast, Cyp11a1f/f;SCCmtp-Cre female mice are fertile. Our findings suggest that the protein levels of P450scc in ovary play a key role in uterus development, ovulation and reproductive capacity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54031 |
全文授權: | 有償授權 |
顯示於系所單位: | 生理學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-104-1.pdf 目前未授權公開取用 | 2.81 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。